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Title page for ETD etd-03282011-101620


Type of Document Master's Thesis
Author Dulaney, Weston Patrick
Author's Email Address wes.dulaney@gmail.com
URN etd-03282011-101620
Title Determining the Mechanism of SV40-induced DNA Damage
Degree Master of Science
Department Biological Sciences
Advisory Committee
Advisor Name Title
Ellen Fanning Committee Co-Chair
Katherine Friedman Committee Co-Chair
Keywords
  • DNA damage and repair
  • genome instability
  • simian virus 40
  • virus entry
Date of Defense 2010-12-20
Availability unrestricted
Abstract
The ability of SV40 to activate the DNA damage response raises the question of whether the virus also induces DNA damage. Broken nuclear DNA in SV40-infected BSC40 cells could be detected at 5 hours post infection (hpi) using Comet and TUNEL assays. DNA breaks were not detected during infections of non-permissive cells. Agents that inhibit intracellular trafficking of SV40 to the endoplasmic reticulum (ER), or redox processes required for uncoating in the ER, prevented DNA breaks. An increase in phosphorylated histone H2AX (γH2AX), a marker of DNA damage signaling, was detected at 5 hpi and the number of γH2AX-positive cells continued to increase during the infection. A greater percentage of γH2AX positive cells were found with increasing viral multiplicity of infection. To test if viral gene expression is required for DNA breaks, SV40 was UV inactivated to prevent early gene expression, without affecting virus uptake or uncoating. Cells infected with UV-inactivated SV40 displayed γH2AX and DNA breaks at a level comparable to that induced by mock-irradiated virus. Because SV40 is uncoated in the ER during entry, we hypothesized that ER stress, disruption of Ca+2 homeostasis, and reactive oxygen species (ROS) could lead to DNA breaks. Consistent with this notion, the antioxidant N-acetyl cysteine (NAC) prevented nuclear DNA fragmentation, but not DNA damage signaling. Future work will clarify how SV40 entry generates host DNA breaks.
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